Antibodies in vertebrates are typically composed of paired heavy (H) and light (L) chains. The first domain of the combined H and L chains, the VH and VL, are more variable in sequence, and this is the portion of the antibody that recognizes and binds to the antigen. The VH and VL domains recognize the antigen as a pair.
The immune repertoire of camelidae (camels, dromedaries and llamas) is unique in that it possesses unusual types of antibodies referred to as heavy-chain antibodies (Hamers, Casterman C. et al., 1993). These antibodies lack light chains and thus their combining sites consist of one domain, termed VHH.
Recombinant VHH single-domain antibodies (sdAbs) provide several advantages over single-chain Fv (scFv) fragments derived from conventional four-chain antibodies. While sdAbs are comparable to their scFv counterparts in terms of affinity, they outperform scFvs in terms of solubility, stability, resistance to aggregation, refoldability, expression yield, and ease of DNA manipulation, library construction and 3-D structural determinations. Many of the aforementioned properties of VHH sdAbs are desired in applications involving antibodies.
However, the non-human nature of VHHs limits their use in human immunotherapy due to immunogenicity. In this respect, human VH and VL sdAbs are ideal candidates for immunotherapy applications because they are expected to be least immunogenic.
Human VHs and VLs, however, are by and large prone to aggregation, a characteristic common to VHs and VLs derived from conventional antibodies (Davies, J. et al., 1994; Tanha, J. et al., 2001; Ward, E. S. et al., 1989). Thus, attempts have been made to obtain monomer human VHs and VLs suitable for antibody applications. Such VHs and VLs have also displayed other useful properties typical of VHHs such as high expression yield, high refoldability and resistance to aggregation. Synthetic libraries built on these VHs and VLs as library scaffolds might serve as a promising source of therapeutic proteins.
Camelization as well as llamination which involves incorporating key solubility residues from camel and llama VHHs, respectively, into human VHs or VLs have been employed to generate monomeric human VHs and VLs. Synthetic sdAb libraries constructed based on these VHs and VLs and generated by CDR randomization were shown to be functional in terms of yielding binders to various antigens (Davies, J. et al., 1995; Tanha, J. et al., 2001).
In another approach, fully human monomeric VHs and VLs were isolated from human synthetic VH and VL libraries without resorting to engineering of the sort mentioned above. In one experiment a monomeric human VH, was discovered when a human VH library was panned against hen egg lysozyme (Jespers, L. et al., 2004b). More recently, a selection method based on reversible unfolding and affinity criteria yielded many monomeric VHs from synthetic human VH libraries (Jespers, L. et al., 2004a). This finding underlined the fact that an appropriate selection method is key to efficient capturing of rare monomer human VHs with desirable biophysical properties.